All eukaryotic organisms possess gene silencing pathways that on one hand function as defense mechanisms against nucleic acid invaders such as viruses, mobile elements and aberrant transcripts, and on the other hand are involved in the regulation of gene expression. RNA interference or RNAi refers to one such pathway and is a post-transcriptional gene silencing mechanism through which gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous cellular transcripts. The discovery that small non-coding RNAs (small interfering RNAs or siRNAs) are the hallmarks of this process was very soon followed by the realization that small non-coding RNAs are involved in regulating eukaryotic gene expression at several different levels, in both the nuclear and cytoplasmic compartments. The interest in Trypanosoma brucei as a model system to study RNAi and related RNA silencing mechanisms stems primarily from its evolutionary position and its biology. Trypanosomatid protozoa represent one of the deepest branches of the eukaryotic lineage and are therefore considered descendants of "ancient" eukaryotes. Thus, understanding the mechanism of RNA silencing in trypanosomes might give us clues to the evolution of this mechanism and to its biological significance. Furthermore, given that in trypanosomes most gene regulation takes place at the post-transcriptional level, there is the possibility that RNA silencing plays a central role in the regulation of gene expression. Indeed, we have provided evidence that one of the biological functions of RNAi in trypanosomes is to silence retroposon transcripts to most likely prevent or reduce retroposition and its deleterious effects on genome integrity. The long-term goal of this proposal is to understand the biological function of small RNAs in T. brucei with the following specific aims: 1. Clone and characterize small non-coding RNAs in T. brucei and investigate whether similar RNAs exist in other kinetoplastidae. 2. Analyze the biological consequences of ablation of RNAi in T. brucei. 3. Investigate the consequences of ablation of the RNAi mechanism on gene expression in T. brucei.